Difference between revisions of "Part:BBa K3018000"

 
 
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<partinfo>BBa_K3018000 short</partinfo>
 
<partinfo>BBa_K3018000 short</partinfo>
  
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This part encodes a bacterial flavin-containing monooxygenase(FMO), which can catalyze the oxidation of indole. We had the codon-optimized for E. coli. You can use this part to produce indigo through indoxyl and leucoindigo in E. coli.  
+
This part encodes a bacterial flavin-containing monooxygenase(bFMO), which can catalyze the oxidation of indole. We had the codon-optimized for E. coli. You can use this part to produce indigo through indoxyl and leucoindigo in E. coli.  
−
Indigo is a hydrophobic blue dye; the expression of functional FMO can be estimated by the color of culture solution. Clones on LB plates are blue.
+
Indigo is a hydrophobic blue dye; the expression of functional FMO can be estimated by the color of culture solution. Clones on LB plates should be blue when it works.
  
 
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<partinfo>BBa_K3018000 parameters</partinfo>
 
<partinfo>BBa_K3018000 parameters</partinfo>
 
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<h1><b>Tongji_China 2019 Characterization</b></h1>
 +
<br/>
 +
All characterizations are performed in plasmid pET-28a(+), strain E. coli BL21(DE3).
 +
<br/>
 +
<b>Function Validation</b>
 +
To validate the function of this part in E. coli BL21(DE3), we transformed pET-28a(+)-bFMO into E. coli BL21(DE3). Bacte and induced with IPTG (final concentration: 0.5mM) . Culture without induction was used as control.
 +
<br/>
 +
[[Image:T--Tongji_China--pET-28a(+)-bFMO_induced_plate.jpg|left|400px|thumb|Dark blue clones on LB plate with IPTG]]
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<br/>
 +
[[Image:T--Tongji_China--indigores.jpg|left|400px|thumb|Validation of function. Tube on the left was induced with IPTG, on the right was not induced as control. This figure shows a significant difference in color between induced group and control group, indicates the production of indigo. The result supports that this part can work in BL21(DE3).]]
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<br/>
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[[Image:T--Tongji_China--indigo_powder.jpg|left|400px|thumb|Indigo powder extracted from culture medium]]
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<br/>
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[[Image:T--Tongji_China--tjindigo.jpg|left|400px|thumb|Tubes containing blue culture medium, induced by IPTG. Tubes organized in the shape of TJ]]
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<br/>
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<br/>
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<div style="clear: both;"></div>
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<h1><b>Tongji-China 2024: The characterization of bFMO produces a blue gradient.</b></h1>
 +
<br/>
 +
We quantitatively characterized the bFMO gene, providing future iGEM teams with characterization conditions and possibilities for indigo color gradients, and offering a broader range of color options for fields such as bioart and fashion.
 +
<br/>
 +
We have chosen E. coli BL21(DE3) as the chassis strain, which is capable of efficiently expressing T7 promoters under IPTG induction. The plasmids we constructed are shown in the following figure. Initially, we selected Kana as the resistance screening gene, 6xHis as the purification tag, and T7 promoter.
 +
<html><img style="float:left;width:400px" src="https://static.igem.wiki/teams/5298/assets/parts/bfmo-1.jpg"></html>
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<div style="clear: both;"></div>
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Figure 1: Design Map of bFMO-pET28a
 +
 +
<br/>
 +
After gene synthesis at Genewiz, we amplified the gene, extracted the plasmid, and transformed it into BL21(DE3). Upon colony formation, we observed color changes, indicating leaky expression.
 +
<div style="clear: both;"></div>
 +
<html><img style="float:left;width:400px" src="https://static.igem.wiki/teams/5298/assets/parts/bfmo-2.png"></html>
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<div style="clear: both;"></div>
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Figure 2: Leaky Expression of Indigo in Solid Medium
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<br/>
 +
To prevent leaky expression, we replaced the T7 promoter with the medium-strength promoter J23110.
 +
<div style="clear: both;"></div>
 +
<html><img style="float:left;width:400px" src="https://static.igem.wiki/teams/5298/assets/parts/bfmo-3.png"></html>
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<div style="clear: both;"></div>
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Figure 3: Promoter J23110 and Design of Primers
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<br/>
 +
After the ligation reaction, we successfully replaced the T7 promoter with the J23110 promoter and obtained monoclones through transformation.
 +
<div style="clear: both;"></div>
 +
<html><img style="float:left;width:400px" src="https://static.igem.wiki/teams/5298/assets/parts/bfmo-4.jpg"></html>
 +
<div style="clear: both;"></div>
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Figure 4: Monoclonal Selection of E. coli after Promoter Replacement through Spread Plating
 +
 +
<br/>
 +
Subsequently, we proceeded to validate the expression in liquid medium. After picking the successfully transformed monoclones, we cultured them in 12ml shake flasks for 12 hours before adding IPTG (final concentration of 0.5mM) and prepared L-tryptophan (final concentration of 5mg/ml) to the culture. The culture was then incubated at 37°C for 18 hours to observe color development. However, the final coloration failed.
 +
<br/>
 +
Addressing the above issues, we devised the following experimental plan improvements based on their ease of verification:
 +
<br/>
 +
1. Directly prepare a tryptophan-containing medium.
 +
<br/>
 +
2. Measure the OD600 growth curves of cultures in liquid media with different substrate concentrations (5.0/2.5/1/0g/L) before adding the inducer.
 +
<br/>
 +
3. After obtaining the growth curves, add IPTG during the early logarithmic growth phase to induce expression.
 +
<br/>
 +
4. Initially, reduce the characterization temperature to 30°C.
 +
<br/>
 +
<div style="clear: both;"></div>
 +
<html><img style="float:left;width:400px" src="https://static.igem.wiki/teams/5298/assets/parts/bfmo-5.jpg"></html>
 +
<div style="clear: both;"></div>
 +
Figure 5: Growth Curve and Indigo Color Development
 +
 +
<br/>
 +
Our constructed plasmid successfully demonstrated indigo coloration in the medium, as evident from the growth curve and the visible color change.
 +
<div style="clear: both;"></div>
 +
<html><img style="float:left;width:400px" src="https://static.igem.wiki/teams/5298/assets/parts/bfmo-6.png"></html>
 +
<div style="clear: both;"></div>
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Figure 6: Expression of Indigo in Liquid LB Medium
 +
 +
<br/>
 +
1. Characterization in Liquid Medium
 +
<div style="clear: both;"></div>
 +
<html><img style="float:left;width:400px" src="https://static.igem.wiki/teams/5298/assets/parts/bfmo-7.png"></html>
 +
<div style="clear: both;"></div>
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Figure 7: Visualization of Changes in Blue Color Over Time in Liquid Medium
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 +
<div style="clear: both;"></div>
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<html><img style="float:left;width:400px" src="https://static.igem.wiki/teams/5298/assets/parts/bfmo-8.png"></html>
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<div style="clear: both;"></div>
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Figure 8: Changes in Solution Purity and Brightness Over Time in Liquid Medium
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<br/>
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2. Characterization in Solid Medium
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<div style="clear: both;"></div>
 +
<html><img style="float:left;width:400px" src="https://static.igem.wiki/teams/5298/assets/parts/bfmo-9.png"></html>
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<div style="clear: both;"></div>
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Figure 9: Box Plot of Univariate Analysis
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<div style="clear: both;"></div>
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<html><img style="float:left;width:300px" src="https://static.igem.wiki/teams/5298/assets/parts/bfmo-10.jpg"></html>
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<div style="clear: both;"></div>

Latest revision as of 13:33, 2 October 2024


Bacterial flavin-containing monooxydase, codon-optimized

This part encodes a bacterial flavin-containing monooxygenase(bFMO), which can catalyze the oxidation of indole. We had the codon-optimized for E. coli. You can use this part to produce indigo through indoxyl and leucoindigo in E. coli. Indigo is a hydrophobic blue dye; the expression of functional FMO can be estimated by the color of culture solution. Clones on LB plates should be blue when it works.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Tongji_China 2019 Characterization


All characterizations are performed in plasmid pET-28a(+), strain E. coli BL21(DE3).
Function Validation To validate the function of this part in E. coli BL21(DE3), we transformed pET-28a(+)-bFMO into E. coli BL21(DE3). Bacte and induced with IPTG (final concentration: 0.5mM) . Culture without induction was used as control.

Dark blue clones on LB plate with IPTG


Validation of function. Tube on the left was induced with IPTG, on the right was not induced as control. This figure shows a significant difference in color between induced group and control group, indicates the production of indigo. The result supports that this part can work in BL21(DE3).


Indigo powder extracted from culture medium


Tubes containing blue culture medium, induced by IPTG. Tubes organized in the shape of TJ



Tongji-China 2024: The characterization of bFMO produces a blue gradient.


We quantitatively characterized the bFMO gene, providing future iGEM teams with characterization conditions and possibilities for indigo color gradients, and offering a broader range of color options for fields such as bioart and fashion.
We have chosen E. coli BL21(DE3) as the chassis strain, which is capable of efficiently expressing T7 promoters under IPTG induction. The plasmids we constructed are shown in the following figure. Initially, we selected Kana as the resistance screening gene, 6xHis as the purification tag, and T7 promoter.

Figure 1: Design Map of bFMO-pET28a


After gene synthesis at Genewiz, we amplified the gene, extracted the plasmid, and transformed it into BL21(DE3). Upon colony formation, we observed color changes, indicating leaky expression.

Figure 2: Leaky Expression of Indigo in Solid Medium


To prevent leaky expression, we replaced the T7 promoter with the medium-strength promoter J23110.

Figure 3: Promoter J23110 and Design of Primers


After the ligation reaction, we successfully replaced the T7 promoter with the J23110 promoter and obtained monoclones through transformation.

Figure 4: Monoclonal Selection of E. coli after Promoter Replacement through Spread Plating


Subsequently, we proceeded to validate the expression in liquid medium. After picking the successfully transformed monoclones, we cultured them in 12ml shake flasks for 12 hours before adding IPTG (final concentration of 0.5mM) and prepared L-tryptophan (final concentration of 5mg/ml) to the culture. The culture was then incubated at 37°C for 18 hours to observe color development. However, the final coloration failed.
Addressing the above issues, we devised the following experimental plan improvements based on their ease of verification:
1. Directly prepare a tryptophan-containing medium.
2. Measure the OD600 growth curves of cultures in liquid media with different substrate concentrations (5.0/2.5/1/0g/L) before adding the inducer.
3. After obtaining the growth curves, add IPTG during the early logarithmic growth phase to induce expression.
4. Initially, reduce the characterization temperature to 30°C.

Figure 5: Growth Curve and Indigo Color Development


Our constructed plasmid successfully demonstrated indigo coloration in the medium, as evident from the growth curve and the visible color change.

Figure 6: Expression of Indigo in Liquid LB Medium


1. Characterization in Liquid Medium

Figure 7: Visualization of Changes in Blue Color Over Time in Liquid Medium

Figure 8: Changes in Solution Purity and Brightness Over Time in Liquid Medium
2. Characterization in Solid Medium

Figure 9: Box Plot of Univariate Analysis